Historically, factors affecting hematopoietic cells have been detected in an assay measuring the proliferation and/or differentiation of bone marrow cells in soft agar cultures. The factors showing this activity have been collectively called colony-stimulating factors (CSFs). More recently, it has been found that a variety of CSFs exist which, in part, can be classified by the hematopoietic lineages that are stimulated.
In human and murine systems, these proteins include G-CSF and M-CSF. These proteins stimulate the in vitro formation of predominantly neutrophilic granulocyte and macrophage colonies, respectively. Interleukin-2 ("IL-2") stimulates the proliferation of both activated T-cells and activated B-cells, but is not considered a colony stimulating factor.
GM-CSF and interleukin-3 ("IL-3", also known as "Multi-CSF") stimulate the formation of macrophage and both neutrophilic and eosinophilic granulocyte colonies. In addition, IL-3 stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies (D. Metcalf "The hematopoietic colony-stimulating factors", 1984, Elsevier, Amsterdam, and D. Metcalf, Science 299 (1985) 16-22).
Growth factor-induced cell proliferation is a complicated process. Following highly specific binding of the growth factor to its receptor at the cell surface, the complex is internalized by endocytosis and induces an intracellular response often preceded by phosphorylation of the receptor (Sibley et al., Cell 48 (1987) 913-922). These intracellular signals result in specific gene transcription and finally in DNA synthesis and cell replication.
There is considerable interest in the CSFs, since they may be therapeutically useful for restoring depressed levels of hematopoietic and lymphoid stem cell-derived cells.
Human IL-3 ("hIL-3") is such a CSF. Mature hIL-3 consists of 133 amino acids; the protein contains one disulfide bridge and has two potential glycosylation sites (Yang et al., Cell 47 (1986) 3-10). It has inter alia the following activities:
1) stimulation of colony formation by human hematopoietic progenitor cells wherein the colonies formed include erythroids, granulocytes, megakaryocytes, granulocyte macrophages, and mixtures thereof; and PA1 2) stimulation of DNA synthesis by human acute myelogenous leukemia (AML) blasts. PA1 Asp.sup.21 Glu.sup.22 .fwdarw.Lys.sup.21 Ar.sup.22 PA1 Asp.sup.36 .fwdarw.Arg.sup.36 PA1 Glu.sup.43 Asp.sup.44 .fwdarw.Lys.sup.43 Arg.sup.44 PA1 Arg.sup.54 Arg.sup.55 .fwdarw.Glu.sup.54 Asp.sup.55 PA1 Asp.sup.46 .fwdarw.Lys.sup.46 or Arg.sup.46 PA1 Glu.sup.50 .fwdarw.Lys.sup.50 or Arg.sup.50, PA1 Glu.sup.59 .fwdarw.Lys.sup.59 or Arg.sup.59 PA1 Glu.sup.59 .fwdarw.Gly.sup.59 or Pro.sup.59, PA1 Ar.sup.63 Ala.sup.64 .fwdarw.Pro.sup.63 Gly.sup.64 PA1 Glu.sup.75 .fwdarw.Arg.sup.75 or Gly.sup.75 PA1 Lys.sup.79 .fwdarw.Glu.sup.79 PA1 Arg.sup.94 .fwdarw.Pro.sup.94 PA1 His.sup.98 Lys.sup.100 Asp.sup.101 .fwdarw.Glu.sup.98 Asp.sup.100 Gln.sup.101 PA1 Glu.sup.106 .fwdarw.Lys.sup.106 PA1 Arg.sup.108 Arg.sup.109 Lys.sup.110 .fwdarw.Glu.sup.108 Asp.sup.109 Glu.sup.110, PA1 Phe.sup.113 Tyr.sup.114 .fwdarw.Ala.sup.113 Thr.sup.114 PA1 Cys.sup.16 .fwdarw.Ala.sup.16 PA1 Cys.sup.84 .fwdarw.Ala.sup.84 PA1 Cys.sup.16 Cys.sup.84 .fwdarw.Ala.sup.16 Ala.sup.84
Useful agonists and antagonists of a protein can be created once the structure-function relationship of the molecule is understood. Generally, this relationship is studied by modifying, replacing or deleting amino acids. In this way, information can be obtained about the importance of each of the amino acids for the activity of the protein. Important domains of proteins may be the active site, metal and cofactor binding sites, receptor binding sites, the amino acids involved in subunit interactions, and the antigenic determinants.
Once the primary sequence of a protein has been determined, various procedures can be employed to study the above-mentioned characteristics. For example, if primary structures of homologous proteins from other species are available, the sequences can be compared. Conserved sequences are often indicative of the importance of certain amino acids.
Secondary structures can be predicted with the use of known algorithms. See, e.g., Hopp and Woods, Proc. Natl. Acad. Sci. USA 78 (1981) 3824-3828, Garnier et al., J. Mol. Biol. 120 (1978) 97-120, Biou et al., Prot. Eng. 2 (1988) 185-191, Carmenes et al., Biochem. Biophys. Res. Commun. 159 (1989) 687-693.
If interspecies homology between homologous proteins is high and the 3-D structure of one of them is known, important amino acids can also be deduced from this structure.
Primary and/or spatial-structure data can be used to make an educated guess for mutagenesis experiments. Expression of mutagenized proteins and the testing of these muteins in biological assays provides information about the relative importance of certain amino acids.
The aim of the present invention is to provide IL-3 mutants with similar or improved pharmaceutical properties with respect to the native IL-3, preferably using the procedures mentioned above.